The pathways of cellular protein degradation and their regulation in eukaryotic cells are poorly understood, and it is unclear whether the transfer of these proteins into lysosomes is an obligatory step. A new approach has been devised which will use highly purified proteins, labeled with iodine-125, and modified by amidation, acylation, and exposure to guanidine hydrochloride. These will constitute a series of substrates which will vary in size, charge, surface properties and conformation. Intrinsic rate constants for the lysosomal degradation of these native and modified proteins will be measured after their uptake by pinocytosis into cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK). These same proteins will also be inserted directly into the extralysosomal-cytoplasmic compartment of these cells. This will be accomplished by encapsulating the proteins in large unilamellar liposomes (LUV) and fusing them with MPM and BHK cells. After fusion, the rates of degradation of these "new" cytoplasmic proteins will be measured. The extent to which these proteins are accumulated in lysosomes will also be determined by isolating the particulate-lysosomal subcellular fractions. Similar data will be obtained in the presence of proteinase inhibitors, inhibitors of energy metabolsm, agents which disrupt the contractile elements in cells, and culture conditions resembling the fed and fasted states. The results will indicate whether a lysosomal pathway is kinetically competent to account for protein turnover and will yield quantitative estimates of the contributions of putative extralysosomal pathways. They will identify properties of proteins which influence their rates and pathways of degradation and will help to characterize the degradative machinery in cells. Consequently, these studies will provide unique information about a central problem in cellular physiology and biochemistry, and will have an important relevancy to medicine. Thus, the development of effective therapies for several inherited and acquired dystrophic disorders, which are characterized by increased rates of tissue protein turnover, can be expected to depend on an improved understanding of the normal pathways of protein catabolism in cells.